1. Field of the Invention
This invention relates generally to the processing of samples such as in the field of separation of biomolecules such as proteins and, in particular, separations by capillary electrophoresis and the use of capillary electrophoresis to detect such biomolecules and in the field of assays in general.
In a range of technology-based business sectors, including the chemical, bioscience, biomedical, and pharmaceutical industries, it has become increasingly desirable to develop capabilities for rapidly and reliably carrying out chemical and biochemical reactions in large numbers using small quantities of samples and reagents. Carrying out a massive screening program manually, for example, can be exceedingly time-consuming, and may be entirely impracticable where only a very small quantity of a key sample or component of the analysis is available, or where a component is very costly.
Accordingly, considerable resources have been directed to developing methods for high-throughput chemical synthesis, screening, and analysis. Subsequently, considerable art has emerged, in part, from such efforts. Automated laboratory workstations have contributed significantly to advances in pharmaceutical drug discovery and genomics over the past decade. See for example, U.S. Pat. Nos. 5,104,621 and 5,356,525 (Beckman Instruments). More specifically, robotics technology has played a major role in providing a practical useful means for enabling high throughput screening (AS) methods. Reference can be made to U.S. Pat. No. 4,965,049. Highly parallel and automated methods for DNA synthesis and sequencing have also contributed significantly to the success of the human genome project to date.
Computerized data handling and analysis systems have also emerged with the commercial availability of high-throughput instrumentation for numerous life sciences research and development applications. Commercial software, including database and data management software, has become routine in order to efficiently handle the large amount of data being generated. Bioinformatics has emerged as an important field.
With the developments outlined above in molecular and cellular biology, combined with advancements in combinatorial chemistry, have come an exponential increase in the number of targets and compounds available for screening. In addition, many new genes and their expressed proteins will be identified by the Human Genome project and will therefore greatly expand the pool of new targets for drug discovery. Subsequently, an unprecedented interest has arisen in the development of more efficient ultra-high throughput methods and instrumentation for pharmaceutical and genomics screening applications.
In recent parallel technological developments, miniaturization of chemical analysis systems, employing semiconductor processing methods, including photolithography and other wafer fabrication techniques borrowed from the microelectronics industry, has attracted increasing attention and has progressed rapidly. The so-called "lab-chip" technology enables sample preparation and analysis to be carried out on-board microfluidic-based cassettes. Moving fluids through a network of interconnecting enclosed microchannels of capillary dimensions is possible using electrokinetic transport methods.
Application of microfluidics technology embodied in the form of analytical devices has many attractive features for pharmaceutical high throughput screening. Advantages of miniaturization include greatly increased throughput and reduced costs, in addition to low consumption of both sample and reagents and system portability. Implementation of these developments in microfluidics and laboratory automation holds great promise for contributing to advancements in life sciences research and development.
Capillary-based separations are widely used for analysis of a variety of analyte species. Numerous subtechniques, all based on electrokinetic-driven separations, have been developed. Capillary electrophoresis is one of the more popular of these techniques and can be considered to encompass a number of related separation techniques such as capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic chromatography. In the context used throughout this application, the phrase "capillary electrophoresis" is used to refer to any and all of the aforementioned electrokinetic separation subtechniques.
Microfluidic devices provide fluidic networks in which biochemical reactions, sample injections and separation of reaction products can be achieved. The application of high voltage to conductive fluids within these channels leads to electroosmotic and/or electrophoretic pumping, providing both mass transport and separation of components within the sample. In these microfluidic devices, fluid flow and reagent mixing is achieved using electrokinetic transport phenomena (electroosmotic and electrophoretic). Electrokinetic transport is controlled by regulating the applied potentials at the terminus of each channel of the microfluidic device. Within the channel network, cross intersections and mixing tees are used for valving and dispensing fluids with high volumetric reproducibility (0.5% RSD). The mixing tee can be used to mix proportionately two fluid streams in ratio from 0 to 100% from either stream simply by varying the relative field strengths in the two channels.
When capillary electrophoresis is carried out using a fluid electrophoretic medium, the medium itself may undergo bulk flow migration through the capillary tube toward one of the electrodes. This electroosmotic flow is due to a charge shielding effect produced at the capillary wall interface. In the case of standard fused silica capillary tubes, which carry negatively charged silane groups, the charge shielding produces a cylindrical "shell" of positively charged ions in the electrophoresis medium near the surface wall. This shell, in turn, causes the bulk flow medium to assume the character of a positively charged column of fluid and migrate toward the cathodic electrode at an electroosmotic flow rate.
In some instances a prerequisite for conducting assays on microfluidic devices is the ability to transport large proteins (positive and negatively charged), substrates, cofactors and inhibitors or test compounds. Electroosmotic pumping has to be used to transport reagents and samples. Therefore, control of the electroosmotic flow (EOF) and capillary wall chemistry is critical to the success of a microfluidic device. It is well recognized that EOF is essential to move oppositely charged molecules in a single run; for example EOF will be used to mix a lest compound (positively charged) with a negatively charged substrate of an enzyme.
Capillary surface modifications have been an area of active research since the introduction of capillary electrophoresis. This has been prompted by the fact that basic solutes and especially proteins undergo adsorption onto the surface of capillaries. The interaction of solute with the capillary wall leads to band-broadening and in some cases irreversible adsorption. There is an enormous amount of literature describing surface modification of capillary surfaces to separate proteins. The adsorption of proteins on the walls of capillaries is a common problem in the analysis of proteins by capillary electrophoresis. Buffer additives, non-covalent coating and covalent coating have been reported to decrease protein adsorption on the walls of capillaries. Covalent coatings are especially useful in protocols that require minimal concentration of organic materials in the electrokinesis buffer. Dynamic coating is more practical to use in studies, in which separation of analyte from buffer is not important such as, for example, analysis of an enzymatic reaction.
Coatings on capillaries can be classified into two groups, one in which the modifications on the surface inhibit EOF while in others the coatings are designed to retain a certain level of EOF.
2. Previous Disclosures
Wiktorowicz in U.S. Pat. No. 5,015,350 (1991) discloses a method for achieving desired electroosmotic flow characteristics in a capillary tube having charged surface groups. An electrolyte solution containing a compound effective to stably alter the charge of the tube walls is drawn into and through the tube while the electroosmotic flow rate in the tube is being monitored, until a desired electroosmotic flow rate is achieved. The method is reported to optimize electrophoretic separation of charged protein or nucleic acid species in a capillary tube and to produce capillary tubes with desired charge density properties. The compound forms a coating on the tube walls and the method is referred to as a flow-rate controlled surface charge coating or FCSC.
U.S. Pat. No. 5,611,903 (Janssens, et al.) discusses capillary electrophoresis method using initialized capillary and polyanion-containing buffer and chemical kit therefor. The initiator forms a first coating on the inner wall of the capillary and the polyanion in the buffer forms a second coating on the first coating. The polyanion is not in the electrophoresis buffer, which contains the sample to be analyzed.
Okafo, et al., describes effective ion pairing for the separation of basic proteins in capillary electrophoresis. A sodium salt of phytic acid (myoinositol hexakis-(dihydrogen phosphate) is added to the separation buffer.
Fu-Tai (U.S. Pat. No. 5,259,939, 1993) describes the use of zwitterionic salts in capillary electrophoresis to prevent protein adsorption to walls.
Hjerten, et al., (Electrophoresis 1993, 14(5-6):390-396) describes the use of methylcellulose and dextrans to coat fused silica capillaries to eliminate EOF and adsorption of proteins to the capillary walls.
Engelhardt, et al. (EP 0 665 430 A1), discuss a capillary made of plastics material for use in capillary electrophoresis and process for its production. In the modified capillary according to Engelhardt the functional groups can be chemically bonded to atoms of the base plastics material. The functional groups can be created from or are directly chemically bonded to sites of the base polymer capillary material which are created in addition to natural occurring active sites of the polymer. Preferably, the functional residues are predominately hydrophilic groups. In particular the functional groups can be hydroxyl groups. Also, in particular, the functional groups can be amino groups or ammonium groups. With particular advantage the functional groups can be of a type and be present in an amount adapted to effect a desired variation in electrokinetic behavior. Preferably, the functional groups are adapted to control the electroosmotic flow (EOF). In the modified polymer capillary according to Engelhardt, the functional groups can be non-ionic and/or ionic. The functional groups can be ionic with positive charge; also, the functional groups can be ionic with negative charge. The functional property of the inner surface of a capillary modified according to the invention is intended to be that proteins do at most only reversibly adhere; preferably, they do not at all adhere thereon.
U.S. Pat. No. 5,391,274 (Chia-Hui Shieh) discloses methods for controlling electroosmotic flow in coated capillary electrophoresis columns. The methods discussed involve varying the concentration of a multi-valent buffer compound in electroosmotic buffer in order to control the electroosmotic flow in capillary columns having interior surfaces coated with charged organic coatings.
Nikiforov, et al., disclose methods and systems for enhanced fluid transport in WO 98/45929. The methods generally comprise providing an effective concentration of at least one zwitterionic compound in the fluid containing the material that is to be transported or directed.
Whitesides, et al. (Anal Chem.1997, 69(7):1370-1379), Regneir, et al., J. Chromatogr. 1990, 516:69-78; Li, et al., J. Chromatogr. 1994, 680:431-435 describe a method to limit adsorption of proteins based on the adsorption of positively charged polymers on the negatively charged inner surface of fused silica capillaries. The positively charged surface did not adsorb positively charged proteins but could promote adsorption of negatively charged proteins and compounds. Other workers in this area include Wiktorowicz, et al., Electrooresis 1990, 11:769-774; Katayama, et al., Anal. Chem. 1998, 70:2254-2260; Danillo Corradini, et al., Electrophoresis 1995, 16:630-635; Capellil, et al., J. Biochem Biophys Methods 1996, 14:32(2), 109-124).
One problem with the above approaches appears, based on our studies, to be that positively charged proteins can be moved by anodal EOF but that negatively charged substrates and cofactors can not be prevented from adsorbing to the modified capillary walls under optimum reaction conditions of kinase.
Rassi, et al. Electrophoresis 1993, 14:396-406) describes multiple covalent polyether coating on capillaries to achieve both switchable anodal and cathodal EOF.
Novotny, et al., (Electrophoresis 1995, 16:396-401) report a versatile, hydrolytically stable coating of fused silica capillaries with acrylamide and cellulose. These capillaries have low EOF and were used to separate peptides, glycoproteins, etc.
A. Fridstrom, et al., PCT WO98/00709 describe adsorbing phenyl dextran to polypropylene capillaries by hydrophobic association. They demonstrated that phenyl dextran modifies the polypropylene surface by making it more hydrophilic and increases the compatibility of polypropylene columns for proteins. A similar disclosure is found in J. Microcolumn Separation 1997, 9:1-7.
Regneir, et al. (Anal Chem. 1993, 6:2655-2662; 1993, 65:2029-2035; 1993, 65:3267-3270; J. Chromatography 1992, 608:217-224) describe a concept in reaction based chemical analysis. By utilization of variability in electrophoretic mobilities among charged species, spatially distinct zones of chemical reagents can be electrophoretically merged under the influence of an applied electric field. Sequential injections of different reagents into capillary are utilized to meter in reagents in the capillary. Reagent with lower electrophoretic mobility (sum of electrophoretic and electroosmotic transport velocities) is metered in first followed by reagents with higher electrophoretic mobility. Mixing occurs when the reagent with higher electrophoretic mobility overtakes the reagent with lower electrophoretic mobility.
Regneir, et al., supra, describe another concept in reaction based chemical analysis. However, this concept does not appear to be applicable to microfluidic devices. One problem is that the electrophoretic mobility of all the components in the reaction need to be known. In high throughput screening applications the electrophoretic mobility of test compounds is unknown. In the above methodology the spatial positioning of the analytes and analytical reagent zones in the capillary is determined by the sign and magnitude of the electrophoretic mobility of the various species involved so that the appropriate reagents approach and engage each other under the influence of an applied electric field. If the electrophoretic mobility of one of the analytes is not known, then determining the sequence of injections of different reagents is not possible. Another problem is that the authors mention that nonspecific binding of proteins to capillary walls is a major problem. Another problem is formation poor plugs (as identified by poor peak shapes or skewed peaks) due to unwanted protein and capillary wall interactions (adsorption-desorption), hydrophobic interactions, and ion-exchange interactions. Another problem appears to be that, when the electrophoretic mobility of two reagents vary substantially, full interpenetrating will be difficult to achieve even under high electric field strengths. For example, the ability to mix a positively charged enzyme with a negatively charged substrate is a problem that has not been addressed by the authors.
G. M. Whitesides, et al. (J. Med. Chem. 1993, 36:126-133; 1992, 35:2915-17;) describe a similar concept. They selected carbonic anhydrase and arylsulfonamides to determine kinetic and equilibrium binding constants. The nonspecific binding of both carbonic anhydrase and arylsulfonamides to capillary walls is limited. Whitesides, et al. (J. Org. Chem. 1993, 58:5508-5512) describe two types of enzyme-catalyzed reactions in electrophoresis capillaries. These reactions illustrate the interplay of the mobilities of the enzyme, substrates and products in the analysis of electropherograms during the reaction in the capillary. Whitesides points out that, to study protein ligand interactions by affinity capillary electrophoresis or enzyme catalyzed reactions in capillaries, the adsorption of protein to the surface of the capillary should be minimum.
Ramsey, et al. Anal. Chem. 1997, 69:3407-3412) demonstrated an enzyme (.beta.-galactosidase) assay within a microfabricated network. The authors used electrophoretic flow to control the dilution of the reagents used in .beta.-galactosidase assay. Even though this paper demonstrates the power of precise metering and mixing of reagents in a microfluidic device, it clearly admits that protein adsorption to channel walls will be a major problem. For example, enzyme and inhibitor dilution is performed by manually adding different concentration of enzymes and inhibitor to the reservoir. Multiple runs on the device is not practical as the background signal due to enzyme adsorption increases from run to run.
Girault, et al. (Anal. Chem. 1997, 69:2035-2042) describe the use of BSA to coat plasma treated microfluidic devices to reduce nonspecific binding of proteins and yet maintain electroosmotic flow. Harrison, et al. (Clin Chem. 1998, 44(3):591-598; Anal. Chem. 1997, 69:1564-1568) describe the use of bovine serum albumin (BSA) to dynamically coat microfluidic devices to reduce nonspecific binding. The apparent problems of dynamic coating with BSA appear to be that BSA does not prevent nonspecific binding completely, that the electroosmotic flow generated by BSA is pH dependent and that positively charged proteins stick to BSA coated surfaces.
Concentration of biological samples on a microliter scale and analysis by capillary electrophoresis is discussed in U.S. Pat. No. 5,766,435 by Liao, et al.